PROJECT SUMMARY/ ABSTRACT HIV infection is effectively controlled for many patients by long-term use of combination therapies that target actively replicating virus. However, currently available therapies do not lead to viral eradication and HIV remains an incurable, chronic condition. For many patients, substance abuse complicates the chronic administration of anti-retrovirals and predisposes to viralogic failure. Vulnerable patients who are unable to consistently use life-long, daily anti-retrovirals urgently need a cure. One promising strategy to eliminate latently infecting cells is ?shock and kill,? a strategy that depends on ?shocking? latent provirus to an active state so that it can be targeted effectively by medications or host immune factors. However, to effectively drug latently infected cells, we need a deeper understanding of the cellular factors that control viral latency. Functional testing of candidate host factors is critical to understand the mechanisms that control HIV latency. However functional genetic studies in primary human immune cells have been largely impossible until recently. We recently developed a robust CRISPR/Cas9-based technology that enables both `knock- out' and `knock-in' genome editing in primary human T cells. We now propose a comprehensive strategy to test how specific host factors control HIV latency in primary immune cells. The central innovation of this proposal is a high-throughput platform for rapidly generating isogenic human T cells with candidate factors deleted in an arrayed fashion. CRISPR/Cas9 ribonucleoproteins (RNPs) will be transiently delivered to T cells by electroporation to permanently delete targeted genes. This programmable platform will be exploited to rapidly identify and characterize human host factors that regulate multiple stages of the HIV lifecycle. We will focus on elucidating the key genes that control HIV latency and re-activation. Functional assessment of candidates will be performed using a novel HIV dual-fluorescence reporter that can differentiate between latently and productively infected primary T cells. The integration of genome-scale analysis and functional genetics in primary human T cells will accelerate the discovery of novel pathways that regulate HIV infection and latency. Importantly, we will validate the function of these novel regulators of latency using blood samples from HIV-infected patients with substance abuse. These studies are designed to develop targets for a new generation of HIV therapeutics aimed at eradicating latent virus from patients.